The active microbial community more accurately reflects the anaerobic digestion process : 16S rRNA (gene) sequencing as a predictive tool

Amplicon sequencing methods targeting the 16S rRNA gene have been used extensively to investigate microbial community composition and dynamics in anaerobic digestion. These methods successfully characterise amplicons, but do not distinguish micro-organisms that are actually responsible for the process. In this research, the archaeal and bacterial community of 48 full-scale anaerobic digestion plants were evaluated on DNA (total community) and RNA (active community) level via 16S rRNA (gene) amplicon sequencing. A significantly higher richness and overall diversity on DNA compared with the RNA level was observed for archaea, but not for bacteria. Beta diversity analysis showed a significant difference in community composition between the DNA and RNA of both bacteria and archaea, yet, the difference was less pronounced for the bacteria. This related with 25.5 and 42.3% of total OTUs for bacteria and archaea, respectively, that showed a significant difference in their DNA and RNA profiles. Similar operational parameters affected the bacterial and archaeal community, yet, the differentiating effect between DNA and RNA was much stronger for archaea. Co-occurrence networks and functional prediction profiling confirmed the clear differentiation between DNA and RNA profiles. The apparent strong difference between the total and active archaeal community, as determined on different community levels, indicates that the active archaeal community reflects a specialized and organized structure. In contrast, the total and active bacterial community showed a similar community structure, however, community composition also more strongly differed between the total and active community. In conclusion, the clear difference between RNA and DNA based community screening confirms the importance of this combined approach to obtain a broad general overview, not only on the total and active community, but also in terms of potential collaboration and competition and predicted functionality. These results can serve as a basis for further integrated process engineering of the anaerobic digestion process